Splice site m6A methylation prevents binding of DGCR8 to suppress KRT4 pre-mRNA splicing in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m6A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m6A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.


INTRODUCTION
Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide, which accounts for 2-3% of all cancers (Ferlay et al., 2010;Hasegawa et al., 2021). It is estimated that there are around 350,000 new cases and 170,000 deaths from OSCC annually (Bray et al., 2018;Yang et al., 2021). Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50% due to high rates of recurrence and metastasis (Bloebaum et al., 2014;Panzarella et al., 2014). Therefore, it is essential to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies.
Keratin 4 (KRT4) is a member of the type II keratin family (Zhang et al., 2018). Previous studies have indicated that KRT4 expression is downregulated in OSCC (Lallemant et al., 2009;Toruner et al., 2004;Ye et al., 2008). Our recent study has also revealed that KRT4 expression is decreased in OSCC cells and KRT4 suppresses autophagy related 4B cysteine peptidase (ATG4B)-mediated autophagy to inhibit OSCC development (Li et al., 2022). Nevertheless, the mechanism downregulating KRT4 mRNA in OSCC remains unknown.
Dysregulation of pre-mRNA splicing would lead to the downregulation of mRNA (Han et al., 2011;Lee & Rio, 2015). Growing evidence has demonstrated that RNA methylation contributes to proper pre-mRNA splicing and subsequent mRNA expression. For instance, N 6 -methyladenosine (m 6 A) reader YTH domain containing 1 (YTHDC1) associates with m 6 A modified pre-mRNA and facilitates exon inclusion during pre-mRNA splicing by recruiting serine and arginine rich splicing factor 3 (SRSF3) whereas blocking SRSF mRNA binding (Xiao et al., 2016). In addition, the m 6 A methylation of 3 spice site in S-adenosylmethionine (SAM) synthetase pre-mRNA prevents RNA splicing through inhibiting binding of splicing factor U2 small nuclear RNA auxiliary factor 1 (U2AF1) to the 3 spice site (Mendel et al., 2021). Yet the role of m 6 A methylation in KRT4 pre-mRNA splicing has not been reported.
DGCR8 microprocessor complex subunit (DGCR8) is an essential splicing factor for microRNA (miRNA) processing (Guo & Wang, 2019;Michlewski & Caceres, 2019). Besides, DGCR8 is also involved in RNA methylation-mediated miRNA processing. A previous study has indicated that m 6 A methylation enhances the binding of DGCR8 and primary miR-19a to facilitate miRNA processing in cardiovascular endothelial cell (Zhang et al., 2020a). Except regulating miRNA splicing, DGCR8 contributes to mRNA processing as well. In mouse embryonic stem cells, DGCR8 interacts with transcription factor 7 like 1 (Tcf7l1) pre-mRNA to promote the splicing of Tcf7l1 pre-mRNA (Cirera-Salinas et al., 2017). However, the role of DGCR8 in RNA methylation-mediated KRT4 pre-mRNA splicing is largely unknown. Therefore, the primary aim of the current study was to investigate the effects of m 6 A methylation and DGCR8 on KRT4 pre-mRNA splicing in OSCC.

Cell culture
Normal oral keratinocytes (NOK) and OSCC cell line HN6 cells were obtained from Cell Bank at the Chinese Academy of Sciences (Shanghai, China) and cultured as previously described (Li et al., 2022).

Cell transfection
Cells were transfected with METTL3, METTL14 or DGCR8 siRNA and siRNA negative control (NC) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as previously described (Li et al., 2022). Then cells were collected for following experiments at 48 h after transfection. Sequences of siRNAs were listed in Table 1.

Quantitative real-time polymerase chain reaction (qRT-PCR)
After RNA extraction and cDNA synthesis, qRT-PCR was performed by the ABI7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using TB Green R Premix Ex Taq TM II (Tli RNaseH Plus) (Takara) as previously described (Li et al., 2022). Sequences of primers used for qRT-PCR were listed in Table 3.

Methylated RNA immunoprecipitation (MeRIP)
NOK and HN6 cells were collected and lysed. Then nucleic acid fragments were interrupted by ultrasound. Next, cell lysate was incubated with 1 µL m 6 A antibody (1:500, #ab208577, Abcam, Cambridge, MA, USA) overnight at 4 • C. Subsequently, m 6 A antibody and methylated RNA fragments were captured by avidin magnetic beads, and the level of methylated RNA was detected by qRT-PCR.

Statistical analysis
Quantitative data of the current study were present as mean ± standard deviation (SD) and statistical differences were analyzed by SPSS 20 software (SPSS Inc., Chicago, IL, USA) as previously described (Li et al., 2022). Besides, P < 0.05 was considered as statistically significant.

m 6 A methylation inhibits the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA in OSCC
Bioinformatics analysis of CLIP-seq data from ENCORI database further found that splicing factor DGCR8 could bind to KRT4 mRNA in cancer cells (Fig. 4A). Furthermore, All these data together suggested that m 6 A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA in OSCC.

DISCUSSION
The current study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m 6 A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m 6 A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA in OSCC, and silence of DGCR8 prohibited intron splicing of KRT4 pre-mRNA in OSCC. Inhibition of intron splicing could lead to exon exclusion and subsequent expression of non-functional proteins. For instance, serine and arginine rich splicing factor 2 (SRSF2) prevents intron splicing to reduce exon 7 inclusion within survival of motor neuron (SMA) mRNA to produce non-functional SMA protein (Cho et al., 2015;Kashima et al., 2007;Moon et al., 2017). Besides, heterogenous ribonucleaoprotein C1 (hnRNP C1) facilitates exon inclusion within Ron mRNA through promoting intron 10 splicing (Moon et al., 2019). However, our recent study has indicated that KRT4 mRNA level is decreased in OSCC (Li et al., 2022). Therefore, inhibition of intron splicing in KRT4 pre-mRNA should not lead to expression of non-functional KRT4 protein in OSCC.
In addition, suppression of intron splicing also results in intron retention (Pendleton et al., 2017). Several studies have demonstrated that intron retention could result in nuclear pre-mRNA degradation. For example, intron retention stimulates nuclear methionine adenosyltransferase 2A (MAT2A) pre-mRNA decay under high S-adenosylmethionine (SAM) condition (Pendleton et al., 2017). Besides, poly(A)-binding protein nuclear 1 (PABPN1) protein negatively modifies its own expression through binding with PABPN1 pre-mRNA to enhance retention of the 3 -terminal intron and induce nuclear PABPN1 pre-mRNA degradation (Bergeron et al., 2015). Thus, inhibition of intron splicing in KRT4 pre-mRNA should result in intron retention and subsequent nuclear KRT4 pre-mRNA degradation in OSCC.
Growing evidence has indicated that m 6 A methylation plays a crucial role in intron retention. A previous study has revealed that METTL16 increases m 6 A level of a hairpin of MAT2A pre-mRNA to facilitate intron retention (Pendleton et al., 2017). By contrast, overexpression of alkB homolog 5 RNA demethylase (ALKBH5), which is a m 6 Ademethylase, enhances intron retention on E6 mRNA of human papillomavirus type 16 (Cui et al., 2022). Nevertheless, the mechanism of m 6 A methylation regulating intron retention on mRNA remains unclear.
A previous study has demonstrated that DGCR8 also regulates pre-mRNA splicing (Cirera-Salinas et al., 2017). Yet the role of DGCR8 in KRT4 pre-mRNA splicing has not been reported. Our results revealed that silence of DGCR8 prohibited intron splicing of KRT4 pre-mRNA in OSCC. Thus, this study uncovered the effect of DGCR8 on KRT4 pre-mRNA splicing for the first time.
DGCR8 also contributes to m 6 A methylation-mediated splicing of primary microRNAs (pri-miRNAs). In mammalian cells, METTL3 promotes m 6 A methylation of pri-miRNAs to enhance the binding of DGCR8 to pri-miRNAs and splicing of pri-miRNAs (Alarcon et al., 2015). By contrast, our data revealed that m 6 A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. Therefore, DGCR8 might exert opposite effects on pre-mRNA splicing and miRNA processing.
A recent study has revealed that genes related to RNA methylation are associated with immunology, gene mutation and survival of OSCC patients (Wu, Tang & Cheng, 2022). Besides, METTL3 facilitates tumorigenesis and metastasis of OSCC by enhancing BMI1 m 6 A methylation (Liu et al., 2020). Moreover, METTL3 promotes OSCC progress by improving m 6 A methylation of protein arginine methyltransferase 5 (PRMT5) and programmed death-ligand 1 (PD-L1) (Ai et al., 2021). Therefore, above studies and our findings together suggest m 6 A methylation should facilitating OSCC progress.
The role of DGCR8 in OSCC has not been report. Nevertheless, two recent studies have indicated that DGCR8 enhances radiosensitive of head and neck squamous cell carcinoma (Long et al., 2021;Zhang et al., 2020b). Thus, previous studies and our results suggested that DGCR8 should play the opposite role of m 6 A methylation in OSCC. More importantly, these studies could further confirm the validity of our findings.
However, there were some limitations of the current study. For example, this study could be strengthened through identifying functional relevance of m 6 A methylation and DGCR8 to the suppression of OSCC cell growth induced by KRT4. Besides, the findings of this study should be confirmed by in vivo experiments. Figure 6 Schematic diagram of molecular mechanisms for the current study. The current study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m 6 A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. In addition, m 6 A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC.

CONCLUSION
In summary, the current study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m 6 A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. In addition, m 6 A methylation suppressed the binding of DGCR8 to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC (Fig. 6). These results revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 81500864) and Guangdong Basic and Applied Basic Research Foundation (Grant No. 2019A1515011203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: